In the production of recombinant proteins from cultures of microorganisms or cell lines, the final production step is the recovery and optionally the concentration of the product of interest. Culture media in which the cells have been grown and which contain secreted proteins, and, in particular, cell lysates containing intracellular proteins of interest also contain, to a greater or lesser extent, other proteins produced by the cells, apart from other contaminants, such as media components, nucleic acids and the like. In order to obtain a purified protein product, it is therefore necessary to separate the protein of interest from other proteins and polypeptides and other impurities in the crude material containing the protein of interest.
It is often difficult to remove protein contaminant comprising domains of the same nature as the polypeptide of interest.
Vitamin K-dependent proteins are distinguished from other proteins by sharing a common structural feature in their amino terminal part of the molecule. The N-terminal of these proteins, also referred to as the Gla-domain, is rich in the unusual amino acid γ-carboxy glutamic acid which is synthesized from glutamate in a Vitamin K-dependent reaction catalysed by the enzyme γ-glutamyl carboxylase. Because of the presence of about 2 to 12 Gla residues, the Gla-domain is characterised by being capable of binding divalent cations such as Ca2+. Upon binding of metal ions, these proteins undergo conformational changes which can be measured by several techniques such as circular dichroism and fluorescence emission.
The discovery of metal induced conformational changes of Gla-containing proteins (Nelsestuen et. al., J. Biol. Chem. 1976; 251, 6886-6893) together with identification of conformation specific polyclonal antibodies (Furie et al., J. Biol. Chem. 1978; 253, 8980-8987) opened the way for the introduction of conformation specific immunoaffinity chromatography. These antibodies could recognise and bind the Gla-domain in the presence of Ca2+ ions but released the protein upon removal of Ca2+ ions using a Ca2+ chelator such as EDTA or citrate.
In the 1980's conformation specific pseudoaffinity chromatography was developed making use of the unique property of Gla containing proteins to undergo metal induced changes in conformation. Pseudoaffinity chromatography differs from the conventional affinity chromatography in that there is no immobilized affinity ligand involved and it is performed on a conventional chromatographic matrix (Yan S. B., J. Mol. Recog. 1996; 9, 211-218). The Gla protein can be adsorbed to an anion exchange material by eliminating divalent metal ions. Subsequently, elution is performed by adding Ca2+ to the elution buffer.
In 1986, Bjørn and Thim reported purification of recombinant Factor VII on an anion exchange material taking advantage of Ca2+-binding property of Gla-domain of Factor VII (Bjørn S. and Thim L., Research Disclosure, 1986, 26960-26962.). Adsorption was achieved in a buffer without Ca2+ and elution of Factor VII was possible using a Ca2+ containing buffer with low ionic strength and under mild conditions.
Yan et al. have used the same principle for the purification of recombinant human Protein C (Yan S. B. et al., Bio/technology. 1990; 8, 655-661).
While the presence of Gla-domain provides an advantage for separation of Gla containing proteins from other proteins, the inventors of present invention observed that similar properties and behaviour of the Gla containing proteins makes it difficult to separate them from each other. Several conformational specific antibodies raised against one Gla proteins show cross reactivity with other Gla proteins (Furie B. and Furie B., J. Biol. Chem. 1979; 254, 9766-9771; Church et al., J. Biol. Chem. 1988; 263, 6259-6267.).
Brown et al. (Brown et al., J. Biol. Chem. 2000; 275, 19795-19802.) have reported monoclonal antibodies specific for Gla residues. These antibodies could recognize all of the Gla proteins tested: Factor VII, Factor IX, Factor II, Protein C, Protein S, GAS-6, bone matrix Gla protein, conantokin G.
Proteins with a GLA-domain comprises, but is not limited to, the following proteins: GAS-6, Protein S, Factor II (Prothrombin), thrombin, Factor X/Xa, Factor IX/IXa, Protein C, Factor VII/VIIa, Protein Z, Transmembrane gamma-carboxyglutamic acid protein 1, Transmembrane gamma-carboxyglutamic acid protein 2, Transmembrane gamma carboxyglutamic acid protein 3, Transmembrane gamma-carboxyglutamic acid protein 4, Matrix Gla protein, and Osteocalcin.
U.S. Pat. No. 5,633,350 describes a method for the separation of Vitamin K dependent proteins from non-Vitamin K dependent accompanying proteins.
The need for efficiently separating a Vitamin K-dependent protein of interest, such as a Gla-domain containing polypeptide of interest, from protein contaminants is a particularly relevant issue when dealing with the purification of such polypeptides produced in cell cultures, because the host cell (which may not be a human cell line) may produce significant amounts of protein contaminants that can cause undesirable immunogenic reactions upon use of the polypeptide.
Thus, an object of the present invention is to provide suitable methods for the reduction or even removal of the content of protein contaminants in compositions comprising a Vitamin K-dependent protein of interest. A further object of the present invention is to provide compositions comprising a Vitamin K-dependent protein of interest with a very low or even negligible content of protein contaminants.